Bordetella pertussis is the causative agent of whooping cough. Vaccines against whooping cough have been available for many years and have been successful in reducing the disease. The vaccines are either whole-cell (wP), based inactivated B. pertussis cells, or acellular (aP), based on purified B. pertussis antigens. The present invention relates to methods for purifying antigens suitable for use in acellular vaccines.
Current aP vaccines include the INFANRIX™ series, PEDIARIX™ and the BOOSTRIX™ series (all from GSK) and TRIPEDIA™, DAPTACEL™ and ADACEL™ (all from Sanofi Pasteur). These include one or more of the following B. pertussis antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), the 69 kDa pertactin protein (p69), and fimbrial agglutinogens (FIM). Using at least the three PT, FHA and p69 antigens is usual.
References 1 & 2 disclose a process for preparing PT and FHA. After cell growth the culture medium contains PT and FHA. These two antigens are adsorbed onto a solid support (e.g. Perlite) and are then selectively eluted. The two antigens are then each further purified by adsorption onto a hydroxyapatite column. The antigens are then inactivated using glutaraldehyde or formaldehyde.
Reference 3 discloses a process for preparing p69. The p69 antigen is precipitated using ammonium sulfate, either before or after perlite adsorption of PT and FHA. It is then further purified using hydroxyapatite and/or Q-Sepharose.
Reference 4 uses a combination of the methods of references 1 and 3 to purify the p69, PT and FHA antigens to give an aP vaccine. A similar procedure is disclosed in reference 5.
The PT and FHA antigens in the INFANRIX™ products are extracted from fermentation broth by adsorption on hydroxyapatite gel and are then further purified by hydrophobic, affinity and size exclusion chromatography. The p69 antigen in the INFANRIX™ products is extracted from the cells by heat treatment and flocculation using barium chloride and is then further purified by ion exchange, hydrophobic and size exclusion chromatography.
Various methods for purifying FHA, p69, PT and FIM antigens are disclosed in reference 6. Calcium ions are added to a B. pertussis culture containing excess phosphate ions, and the methods are based on the differential adsorption of the various antigens to the calcium phosphate that is formed in situ.
It is an object of the invention to provide further and improved methods for purifying p69 from Bordetella pertussis, to provide an antigen for manufacturing acellular whooping cough vaccines.